Diversity Analysis Reveals Effects of Glycosylated Biotherapeutics
2School of Mathematical Sciences, Tel Aviv University
3Department of Chemistry, University of California, Davis, California
4Institute for Cereal Crops Research, Tel Aviv University
Inflammatory Bowel Disease (IBD) is a chronic illness commonly treated with anti-TNFα glycosylated monoclonal antibodies (mAbs; e.g. Infliximab, Adalimumab, Golimumab), but some patients do not respond well or lose response over time. Sialic acids cover the tips of glycan chains, particularly with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the two most common in mammals. However, humans cannot synthesize Neu5Gc, but can acquire it through red meat diet resulting in induction of serum anti-Neu5Gc antibodies (IgG, IgA, IgM). We hypothesize that Neu5Gc-glycosylation on drugs may further induce anti-Neu5Gc antibody responses in patients, and also promote drug recognition by these antibodies potentially affecting drug efficacy in patients. We investigated 70 pediatric IBD patients treated with Neu5Gc-glycosyated Infliximab over time from the IBD Plexus biobank (Chron’s and Colitis Foundation Association; CCFA). Plasma samples were obtained before therapy and with up to 3-years follow-up for each patient, in total 218 samples. IgG reactivity was examined by glycan microarrays fabricated with 26 Neu5Gc-glycans and their matching Neu5Ac-glycans pairs differing by a single oxygen atom (total 52 glycans) and these were subject to diversity analysis for evaluation of anti-drug immune responses. α-Diversity measures of anti-Neu5Gc IgG decreased across 36 months of treatment and correlations with specific anti-Neu5Gc IgG highlighted anti-Neu5Gc#24 IgG, which presented a 10% increase per year in a mixed model. Furthermore, there was a significant increase in anti-Neu5Ac#23 IgG (Wilcoxon signed-rank test p-value <0.001). After the 3-year therapy (36M), IgG profiles separated into three distinct groups via cluster analysis. Decreased α-diversity measures and β-diversity analysis on obtained groups showed a narrowing IgG repertoire, suggesting a more targeted immune response towards specific antigens. Altogether, these data support altered IgG repertoire after long-term therapy with Infliximab, with a particular increase in anti-Neu5Gc#24 IgG and anti-Neu5Ac#23 IgG. While Neu5Gc is a foreign sugar in humans, Neu5Ac covers all human cells, and glycan 23 in particular is abundant in intestine, as well as in brain and other vital organs. Therefore, increase in specific anti-Neu5Ac#23 IgG indicates a shift in the immune response towards targeting ‘self‘ glycans under Infliximab, possibly mediating autoimmune disease complications, and warranting further investigation.